Persistent Luminescence Strontium Aluminate Nanoparticles as Reporters in Lateral Flow Assays

Demand for highly sensitive, robust diagnostics and environmental monitoring methods has led to extensive research in improving reporter technologies. Inorganic phosphorescent materials exhibiting persistent luminescence are commonly found in electroluminescent displays and glowing paints but are not widely used as reporters in diagnostic assays. Persistent luminescence nanoparticles (PLNPs) offer advantages over conventional photoluminescent probes, including the potential for enhanced sensitivity by collecting time-resolved measurements or images with decreased background autofluorescence while eliminating the need for expensive optical hardware, superior resistance to photobleaching, amenability to quantitation, and facile bioconjugation schemes. We isolated rare-earth doped strontium aluminate PLNPs from larger-particle commercial materials by wet milling and differential sedimentation and water-stabilized the particles by silica encapsulation using a modified Stöber process. Surface treatment with aldehyde silane followed by reductive amination with heterobifunctional amine-poly­(ethylene glycol)-carboxyl allowed covalent attachment of proteins to the particles using standard carbodiimide chemistry. NeutrAvidin PLNPs were used in lateral flow assays (LFAs) with biotinylated lysozyme as a model analyte in buffer and monoclonal anti-lysozyme HyHEL-5 antibodies at the test line. Preliminary experiments revealed a limit of detection below 100 pg/mL using the NeutrAvidin PLNPs, which was approximately an order of magnitude more sensitive than colloidal gold

Microretroreflector-Sedimentation Immunoassays for Pathogen Detection

Point-of-care detection of pathogens is medically valuable but poses challenging trade-offs between instrument complexity and clinical and analytical sensitivity. Here we introduce a diagnostic platform utilizing lithographically fabricated micron-scale forms of cubic retroreflectors, arguably one of the most optically detectable human artifacts, as reporter labels for use in sensitive immunoassays. We demonstrate the applicability of this novel optical label in a simple assay format in which retroreflector cubes are first mixed with the sample. The cubes are then allowed to settle onto an immuno-capture surface, followed by inversion for gravity-driven removal of nonspecifically bound cubes. Cubes bridged to the capture surface by the analyte are detected using inexpensive, low-numerical aperture optics. For model bacterial and viral pathogens, sensitivity in 10% human serum was found to be 10⁴ bacterial cells/mL and 10⁴ virus particles/mL, consistent with clinical utility

HSV-2 PLNP LFA receiver operating characteristic (ROC) curves.

Each panel member was tested and analyzed in replicate (n = 2), by SeraCare, Inc, using HerpeSelect 2 ELISA from Focus Diagnostics, and the resulting absorbance mean was normalized to generate signal-to-cutoff ratios (reactivity ratios). Following SeraCare’s reported specifications, we considered panel members with average reactivity ratios < 1.0 and ≥1.0 as negative and positive, respectively, for human IgG antibodies specific to HSV-2. The panel was tested three times using the HSV-2 PLNP LFA (n = 63) with smartphone-based imaging and app readout. TLs above or below the range of quantitation of the iPhone app were given a value of 71 (the upper limit of quantitation) or 0, respectively. An HSV-2 PLNP LFA receiver operating curve (solid dark blue curve) was generated in MedCalc (version 18.2.1) using test line signal and positive cutoff values, to determine a test positive or negative. Each positive or negative test was then defined as true positive, true negative, false positive, or false negative when referenced to the gold standard method, HerpeSelect 2 ELISA. Based on the ROC analysis, a positive cutoff value of 8 for the TL of HSV-2 PLNP LFA produced the highest sensitivity (96.7%) while maintaining 100% specificity. The ROC curve and analysis gave a value of 0.997 for the area under the curve (AUC) with a 95% confidence interval of 0.937 and 1.00. The confidence region for the ROC curve is represented by the area between the light blue dotted lines.</p

Comparison of single measure intraclass correlations (ICC) in a two-way mixed-effects model for consistency of agreement using different combinations of imaging devices and image analysis tools (method A, B, C) for the same set of LFA strips.

For each method, the ICC is followed by the 95 percent confidence interval enclosed in brackets.</p

Comparison of test metrics for HSV- 2 rapid tests based on the Seracare panel PTH202.

The results for each assay are based on the average of 2 or 3 replicates.</p

Representative images of the HSV PTH202 serum/plasma panel tested with the HSV-2 PLNP LFA.

The strips were imaged as a group on the FluorChem Platform (left side, panel members 1 to 21) or individually on the iPhone platform (right, panel member 2). The images capture a region that encompasses both the test line (TL) and control line (CL) on the test strip. Panel member 16 consistently showed a significantly weaker control line which was undetectable by the smartphone. For further investigation, Panel member 16 was re-tested at a higher dilution (20-fold instead of 3.5-fold), and the picture was added to the panel image (denoted by the asterisk).</p

HSV-2 PLNP LFA schematic.

A sample is diluted and then mixed with PLNPs functionalized with goat anti-human IgGs to form human IgG-PLNP complexes that were dispensed onto the sample pad of the LFA strip. The anti-HSV human IgG PLNP complexes migrated up the membrane and were captured by recombinant HSV gG2 immobilized at the test line. The remaining uncaptured human IgG-PLNP complexes, whether specific to HSV2 gG2 or not, continued further up the strip until they were captured by goat anti-human IgGs immobilized at the control line.</p